Most carotenogenic bacteria synthesize β-carotene from the precursor geranylgeranyl pyrophosphate (GGPP) in three enzymatic steps (PS, PD, and LC; see FIG. 1), and these enzymes are encoded by three separate and distinct genes. There are reports in the literature of single genes in certain filamentous fungi encoding bi-functional enzymes for PS and LC activities (Verdoes, J. C., et al. Mol. Gen. Genet. 262, 453-461 (1999); Velayos, A. et al. Eur. J. Biochem. 267, 5509-5519 (2000); Arrach, N. et al. Proc. Natl. Acad. Sci. USA 98, 1687-1692 (2001); Arrach, N. et al. Mol. Genet. Genomics 266, 914-921 (2002)). In plants and some bacteria, the biochemical conversion of phytoene to lycopene is carried out by two separate enzymes encoded by two separate genes: a phytoene dehydrogenase that converts phytoene only to ζ-carotene and a ζ-carotene dehydrogenase that converts ζ-carotene to lycopene. Additionally, plants require a carotenoid isomerase for this conversion.
Globally, many people suffer from eye dysfunction caused by low vitamin A levels in their diets. In recent years, several research groups have engineered crop plants for the production of carotenoids, and principally β-carotene for its pro-vitamin A activity in an attempt to ultimately provide this vitamin in staple foods. For example, published work (e.g., Shewmaker et al., Plant J., 20, 401, (1999)) demonstrated that expression of a bacterial phytoene synthase (PS) in developing canola seed resulted in a significant increase in carotenoid production in those seeds. As another example, rice endosperm required the expression of phytoene dehydrogenase (PD) and phytoene synthase (PS) activity for β-carotene accumulation (Beyer et al., J. Nutri. 132, 506S, (2002)). The genes for these enzymes were from different biological sources. However, anecdotal information has suggested that subsequent breeding of these rice strains has lead to segregation of the PD and PS genes. These developmental problems are a block to the effective use of transformed plants to produce carotenoids.
The discovery of new enzymes in the carotenoid synthase pathway, and particularly, of enzymes that contain multiple enzyme functions, is therefore desirable for use in genetic engineering of microorganisms and plants for the production of carotenoids by a biosynthetic method.